Chemistry 121(01) Winter 2009-10
Introduction to Organic Chemistry and Biochemistry
Instructor Dr. Upali Siriwardane (Ph.D. Ohio State)
E-mail: upali@chem.latech.edu
Office: 311 Carson Taylor Hall ; Phone: 318-257-4941;
Office Hours: MWF 8:00 am - 10:00 am;
TT 9:00 – 10:00 am & 1:00-2:00 pm.
December 18, 2009 Test 1 (Chapters 12-13)
January 20, 2010 Test 2 (Chapters 14,15 & 16)
February 8, 2010 Test 3(Chapters 17, 18 & 19)
March 1, 2010
Test 4 (Chapters 20, 21 & 22)
March 2, 2010
Comprehensive Make Up Exam:
Chemistry 121 Winter 2010 LA Tech
Chapter 22-1
Human egg and sperm.
Chapter 22. Nucleic Acids
Sections
Chemistry 121 Winter 2010 LA Tech
Chapter 22-2
Chapter 22. Nucleic Acids-Sections
22.1 Types of Nucleic Acids
22.2 Nucleotides: Building Blocks of Nucleic Acids
22.3 Primary Nucleic Acid Structure
22.4 The DNA Double Helix
22.5 Replication of DNA Molecules
22.6 Overview of Protein Synthesis
22.7 Ribonucleic Acids
Chemistry at a Glance: DNA Replication
22.8 Transcription: RNA Synthesis
22.9 The Genetic Code
22.10 Anticodons and tRNA Molecules
22.11 Translation: Protein Synthesis
22.12 Mutations
Chemistry at a Glance: Protein Synthesis
22.13 Nucleic Acids and Viruses
22.14 Recombinant DNA and Genetic Engineering
22.15 The Polymerase Chain Reaction
22.16 DNA Sequencing
Chemistry 121 Winter 2010 LA Tech
Chapter 22-3
Why Nucleic Acid is Important to life?
•
•
•
•
•
•
•
Cells in an organism are exact replicas
Cells have information on how to make new cells
Molecules responsible for such information are nucleic
acids
• Found in nucleus and are acidic in nature
A nucleic acid is a polymer in which the monomer units
are nucleotides.
Two Types of Nucleic Acids:
DNA: Deoxyribonucleic Acid: Found within cell nucleus
• Storage and transfer of genetic information
• Passed from one cell to other during cell division
RNA: Ribonucleic Acid: Occurs in all parts of cell
• Primary function is to synthesize the proteins
Chemistry 121 Winter 2010 LA Tech
Chapter 22-4
Nucleic Acids
• Nucleic Acids: Polymers in which repeating
unit is nucleotide
• A Nucleotide has three components:
• Pentose Sugar: Monosaccharide
• Phosphate Group (PO43-)
• Heterocyclic Base
Base
Phosphate
Chemistry 121 Winter 2010 LA Tech
Sugar
Chapter 22-5
Pentose Sugar
• Ribose is present in RNA and 2-deoxyribose
is present in DNA
• Structural difference:
• a —OH group present on carbon 2’ in ribose
• a —H atom in 2-deoxyribose
• RNA and DNA differ in the identity of the
sugar unit in their nucleotides.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-6
Nitrogen-Containing Heterocyclic
Bases
• There are a total five bases (four of them in
most of DNA and RNAs)
• Three pyrimidine derivatives - thymine (T),
cytosine (C), and uracil (U)
• Two purine derivatives - adenine (A) and
guanine (G)
• Adenine (A), guanine (G), and cytosine (C) are
found in both DNA and RNA.
• Uracil (U): found only in RNA
• Thymine (T) found only in DNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-7
Phospate
• Phosphate - third component of a nucleotide,
is derived from phosphoric acid (H3PO4)
• Under cellular pH conditions, the phosphoric
acid is fully dissociated to give a hydrogen
phosphate ion (HPO42-)
Chemistry 121 Winter 2010 LA Tech
Chapter 22-8
Nucelotide Formation
• The formation of a nucleotide from sugar,
base, and phosphate is visualized below.
• Phosphate attached to C-5’ and base is attached
to C-1’ position of pentose
Chemistry 121 Winter 2010 LA Tech
Chapter 22-9
Nucleotide Nomenclature
Chemistry 121 Winter 2010 LA Tech
Chapter 22-10
Backbone structure for nucleic acid
(a) The generalized
structure of a nucleic
acid. (b) The specific
backbone structure
for a
deoxyribonucleic acid
(DNA). (c) The
specific backbone
structure for a
ribonucleic acid
(RNA).
Chemistry 121 Winter 2010 LA Tech
Chapter 22-11
Adenine, a nucleic acid base
Molecule of Adenine, a
nitrogen-containing
heterocyclic base present
in both RNA and DNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-12
Nucleic acid bases
Two purine bases and
three pyrimidine
bases are found in
the nucleotides
present in nucleic
acids.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-13
Nucleic acid bases
Chemistry 121 Winter 2010 LA Tech
Chapter 22-14
Nucleotides
Table 22.1
Chemistry 121 Winter 2010 LA Tech
Chapter 22-15
Lipids cont’d
Fig. 22.3
The general structure of a nucleic acid in terms of
nucleotide subunits.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-16
DNA Fragment
A four-nucleotidelong segment of DNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-17
Chromosomes
•
•
•
Upon DNA replication the large DNA molecules interacts with
histone proteins to fold long DNA molecules.
The histone–DNA complexes are called chromosomes:
• A chromosome is about 15% by mass DNA and 85% by
mass protein.
• Cells of different kinds of organisms have different
numbers of chromosomes.
• Example: Number of chromosomes in a human cell 46, a
mosquito 6, a frog 26, a dog 78, and a turkey 82
Chromosomes occur in matched (homologous) pairs.
• Example: The 46 chromosomes of a human cell constitute
23 homologous pairs
Chemistry 121 Winter 2010 LA Tech
Chapter 22-18
Protiens and DNA Comparison
A comparison of the primary structures of nucleic acids
and proteins.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-19
DNA double helix
A schematic drawing
of the DNA double
helix that emphasizes
the hydrogen
bonding between
bases on the two
chains.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-20
Hydrogen bonding in Base Pairs
Hydrogen bonding
possibilities
Chemistry 121 Winter 2010 LA Tech
Chapter 22-21
DNA replication
Chemistry 121 Winter 2010 LA Tech
Chapter 22-22
DNA Replication
One strand of DNA grows continuously in the direction of
the unwinding, and the other grows in the opposite
direction.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-23
DNA replication at multiple sites
DNA replication usually occurs at multiple sites within a molecule,
and the replication is bidirectional from these sites.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-24
What in Common Twins Have?
Identical twins share
identical physical
characteristics
because they
received identical
DNA from their
parents.
© Erica Stone / Peter Arnold, Inc.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-25
DNA replication… cont’d
Chemistry 121 Winter 2010 LA Tech
Chapter 22-26
• Protein synthesis is directly under the
direction of DNA
• Proteins are responsible for the formation of
skin, hair, enzymes, hormones, and so on
• Protein synthesis can be divided into two
phases.
• Transcription – A process by which DNA directs
the synthesis of mRNA molecules
• Translation – a process in which mRNA
isdeciphered to synthesize a protein molecule
Transcription
DNA
Chemistry 121 Winter 2010 LA Tech
RNA
Translation
Protein
Chapter 22-27
Differences Between RNA and DNA
Molecules
• The sugar unit in the backbone of RNA is
ribose; it is deoxyribose in DNA.
• The base thymine found in DNA is replaced
by uracil in RNA
• RNA is a single-stranded molecule; DNA is
double-stranded (double helix)
• RNA molecules are much smaller than DNA
molecules, ranging from 75 nucleotides to a
few thousand nucleotides
Chemistry 121 Winter 2010 LA Tech
Chapter 22-28
Types of RNA Molecules
•
•
•
•
•
Heterogeneous nuclear RNA (hnRNA): Formed directly by DNA
transcription.
Post-transcription processing converts the hnRNA to mRNA
Messenger RNA: Carries instructions for protein synthesis
(genetic information) from DNA
• The molecular mass of mRNA varies with the length of the
protein
Small nuclear RNA: Facilitates the conversion of hnRNA to
mRNA.
• Contains from 100 to 200 nucleotides
Ribosomal RNA (rRNA): Combines with specific proteins to
form ribosomes - the physical site for protein synthesis
Ribosomes have molecular masses on the order of 3 million
Chemistry 121 Winter 2010 LA Tech
Chapter 22-29
Types of RNA Molecules
• Transfer RNA (tRNA): Delivers amino acids
to the sites for protein synthesis
• tRNAs are the smallest (75–90 nucleotide units)
Chemistry 121 Winter 2010 LA Tech
Chapter 22-30
Transcription
• Transcription: A process by which DNA
directs the synthesis of mRNA molecules
• Two-step process - (1) synthesis of hnRNA and (2)
editing to yield mRNA molecule
• Gene: A segment of a DNA base sequence
responsible for the production of a specific
hnRNA/mRNA molecule
• Most human genes are ~1000–3500 nucleotide
units long
• Genome: All of the genetic material (the total
DNA) contained in the chromosomes of an
organism
• Human genome is about 20,000–25,000 genes
Chemistry 121 Winter 2010 LA Tech
Chapter 22-31
Steps in the Transcription Process
•
Unwinding of DNA double helix to expose some
bases (a gene):
• The unwinding process is governed by RNA polymerase
•
Alignment of free ribonucleotides along the exposed
DNA strand (template) forming new base pairs
• RNA polymerase catalyzes the linkage of
ribonucleotides one by one to form mRNA molecule
• Transcription ends when the RNA polymerase
enzyme encounters a stop signal on the DNA
template:
• The newly formed RNA molecule and the RNA polymerase
enzyme are released
Chemistry 121 Winter 2010 LA Tech
Chapter 22-32
Post-Transcription Processing:
Formation of mRNA
• Involves conversion of hnRNA to mRNA
• Splicing: Excision of introns and joining of exons
• Exon - a gene segment that codes for genetic information
• Intron – a DNA segments that interrupt a genetic message
• The splicing process is driven by snRNA
• Alternative splicing - A process by which several
different protein variants are produced from a single
gene
• The process involves excision of one or more exons
Chemistry 121 Winter 2010 LA Tech
Chapter 22-33
Exons and Introns of RNA
Heterogenous nuclear RNA contains both exons and introns.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-34
Transcriptome
• Transcriptome: All of the mRNA molecules
that can be generated from the genetic
material in a genome.
• Transcriptome is different from a genome
• Responsible for the biochemical complexity
created by splice variants obtained by hnRNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-35
RNA hairpin loop
A hairpin loop is produced when a single-stranded
RNA doubles back on itself and complementary
base pairing occurs.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-36
Classification of RNA
According to the function of RNA, it can be classified
as:
Messenger RNA: (m-RNA) synthesized on chromosome
and carries genetic information to the ribosomes for
protein synthesis. It has short half-life.
Transfer RNA (t-RNA) is a relatively small and stable
molecule that carries a specific amino acid from the
cytoplasm to the site of protein synthesis on
ribosomes.
Ribosomal RNA (r-RNA) is the major component of
ribosomes, constituting nearly 65%. r-RNA is
responsible for protein synthesis.
Ribozymes are RNA molecules that have catalytic
properties.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-37
Types of RNA
Chemistry 121 Winter 2010 LA Tech
Chapter 22-38
Transcription of DNA to form RNA
The transcription of DNA to form RNA involves an unwinding
of a portion of the DNA double helix.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-39
Exons and Introns of RNA cont’d
An hnRNA molecule containing four exons.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-40
Codes for Amino Acids
Chemistry 121 Winter 2010 LA Tech
Chapter 22-41
tRNA molecule
A tRNA molecule
Chemistry 121 Winter 2010 LA Tech
Chapter 22-42
Aminoacyl-tRNA synthetase
An aminoacyl-tRNA synthetase has an active site for tRNA and a
binding site for the particular amino acid that is to be attached to
that tRNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-43
Anticodon and Codon
The interaction
between anticodon an
codon.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-44
Ribosome Structure
Ribosomes have structures that contain two subunits.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-45
Protein Synthesis: Initiation
Initiation of protein synthesis begins with the formation of an
initiation complex.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-46
Protein Synthesis: Translation
The process of translation that occurs during protein synthesis.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-47
Effects of Antobiotics
Chemistry 121 Winter 2010 LA Tech
Chapter 22-48
Polysome
Several ribosomes can simultaneously proceed along a single
strand of mRNA. Such a complex of mRNA and ribosomes is
called a polysome.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-49
Protein Synthesis Summary
Chemistry 121 Winter 2010 LA Tech
Chapter 22-50
Influenza virus.
Image of an influenza
virus.
NIBSC / SPL / Photo Researchers
Chemistry 121 Winter 2010 LA Tech
Chapter 22-51
Recombinant DNA
Recombinant DNA is made by inserting a gene obtained from DNA of
one organism into the DNA from another kind of organism.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-52
Cleaving DNA patterns using restriction enzymes
Cleavage patterns resulting from the use of a restriction enzyme that
cleaves DNA between G and A bases.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-53
“sticky ends” of recombnants
The “sticky ends” of the cut plasmid and the gene are
complementary and combine to form recombinant DNA.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-54
Polymerase chain reaction process
Chemistry 121 Winter 2010 LA Tech
Chapter 22-55
Polymerase chain reaction process
Chemistry 121 Winter 2010 LA Tech
Chapter 22-56
DNA sequencing
Selected steps in the DNA sequencing procedure for the 10-base DNA
segment 5’ AGCAGCTGGT 3’.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-57
Summary of Nucleic Acids
Nucleotides are basic units of nucleic acids DNA and
RNA.
Nucleotides include pentose, base and phosphoric acid.
Bases include purine or pyrimidine.
Two major purines present in nucleotides are adenine (A)
and guanine (G), and three major pyrimidines are
thymine (T), cytosine (C) and uracil (U).
Ribonucleotides
- adenosine triphosphate (ATP) stores energy.
- NAD and NADP are important carriers of reducing
power.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-58
Summary of Nucleic Acids
DNA
DNA contains genetic information.
DNA contains adenine (A) and guanine (G), and
thymine (T), and cytosine (C). A-T G-C
DNA has a double helical structure.
The bases in DNA carry the genetic
information.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-59
Summary of Nucleic Acids
RNA
• RNA functions as genetic information-carrying
intermediates in protein synthesis.
• It contains adenine (A) and guanine (G), and cytosine
(C) and uracil (U).
• m-RNA carries genetic information from DNA to the
ribosomes for protein synthesis.
• t-RNA transfers amino acid to the site of protein
synthesis
•
r-RNA is for protein synthesis.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-60
Summary of Cell Construction
Biopolymers
protein
Carbohydrates
(polysaccharides)
DNA
RNA
lipids
subunit
bonds for
subunit
linkage
functions
Characteristic
three-D
structure
Chemistry 121 Winter 2010 LA Tech
Chapter 22-61
Primary Structure
• A ribonucleic acid (RNA) is a nucleotide
polymer in which each of the monomers
contains ribose, a phosphate group, and one
of the heterocyclic bases adenine, cytosine,
guanine, or uracil
• A deoxyribonucleic acid (DNA) is a
nucleotide polymer in which each of the
monomers contains deoxyribose, a
phosphate group, and one of the
heterocyclic bases adenine, cytosine,
guanine, or thymine.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-62
Primary Structure
• Structure: Sequence of
•
•
•
•
nucleotides in DNA or
RNA
Primary structure is
due to changes in the
bases
Phosphodiester bond
at 3’ and 5’ position
5’ end has free
phosphate and 3’ end
has a free OH group
Sequence of bases
read from 5’ to 3’
Chemistry 121 Winter 2010 LA Tech
Chapter 22-63
Comparison of the General Primary Structures of
Nucleic Acids and Proteins
• Backbone: -Phosphate-Sugar- Nucleic acids
• Backbone: -Peptide bonds - Proteins
Chemistry 121 Winter 2010 LA Tech
Chapter 22-64
• Nucleic acids have secondary and tertiary structure
• The secondary structure involves two polynucleotide
chains coiled around each other in a helical fashion
• The poly nucleotides run anti-parallel (opposite
directions) to each other, i.e., 5’ - 3’ and 3’ - 5’
• The bases are located at the center and hydrogen
bonded (A=T and GΞC)
• Base composition: %A = %T and %C = %G)
• Example: Human DNA contains 30% adenine, 30% thymine,
20% guanine and 20% cytocine
Chemistry 121 Winter 2010 LA Tech
Chapter 22-65
•
•
•
•
•
•
•
DNA Sequence: the sequence of bases on one polynucleotide
is complementary to the other polynucleotide
Complementary bases are pairs of bases in a nucleic acid
structure that can hydrogen-bond to each other.
Complementary DNA strands are strands of DNA in a double
helix with base pairing such that each base is located opposite
its complementary base.
Example :
List of bases in sequential order in the direction from the 5’ end
to 3’ end of the segment:
5’-A-A-G-C-T-A-G-C-T-T-A-C-T-3’
Complementary strand of this sequence will be:
3’-T-T-C-G-A-T-C-G-A-A-T-G-A-5’
Chemistry 121 Winter 2010 LA Tech
Chapter 22-66
Base Pairing
• One small and one large base can fit inside
the DNA strands:
• Hydrogen bonding is stronger with A-T and G-C
• A-T and G-C are called complementary bases
Chemistry 121 Winter 2010 LA Tech
Chapter 22-67
Practice Exercise
• Predict the sequence of bases in the DNA
strand complementary to the single DNA
strand shown below:
5’ A–A–T–G–C–A–G–C–T 3’
Answer:
3’ T–T–A–C–G–T–C–G–A 5’
Chemistry 121 Winter 2010 LA Tech
Chapter 22-68
• Replication: Process by which DNA
molecules produce exact duplicates of
themselves
• Old strands act as templates for the
synthesis of new strands
• DNA polymerase checks the correct base
pairing and catalyzes the formation of
phosphodiester linkages
• The newly synthesized DNA has one new
DNA strand and old DNA strand
Chemistry 121 Winter 2010 LA Tech
Chapter 22-69
•
•
•
•
•
•
•
DNA polymerase enzyme can only function in the 5’-to-3’
direction
Therefore one strand (top; leading strand ) grows continuously
in the direction of unwinding
The lagging strand grows in segments (Okazaki fragments) in
the opposite direction
The segments are latter connected by DNA ligase
DNA replication usually occurs at multiple sites within a
molecule (origin of replication)
DNA replication is bidirectional from these sites (replication
forks)
Multiple-site replication enables rapid DNA synthesis
Chemistry 121 Winter 2010 LA Tech
Chapter 22-70
Characteristics of Genetic Code
•
The genetic code is highly degenerate:
•
•
•
•
•
•
Many amino acids are designated by more than one codon.
Arg, Leu, and Ser - represented by six codons.
Most other amino acids - represented by two codons
Met and Trp - have only a single codon.
Codons that specify the same amino acid are called synonyms
There is a pattern to the arrangement of synonyms in the
genetic code table.
•
•
•
All synonyms for an amino acid fall within a single box in unless
there are more than four synonyms
The significance of the “single box” pattern - the first two bases
are the same
For example, the four synonyms for Proline - CCU, CCC, CCA, and
CCG.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-71
Characteristics of Genetic Code
• The genetic code is almost universal:
• With minor exceptions the code is the same in all
organisms
• The same codon specifies the same amino acid
whether the cell is a bacterial cell, a corn plant
cell, or a human cell.
• An initiation codon exists:
• The existence of “stop” codons (UAG, UAA, and
UGA) suggests the existence of “start” codons.
• The codon - coding for the amino acid methionine
(AUG) functions as initiation codon.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-72
Practice Exercise
Answers:
a. 3’ GCG–GCA–UCA–ACC–GGG–
CCU–CCU 5’
b. 3’ GCG–ACC–CCU–CCU 5’
Chemistry 121 Winter 2010 LA Tech
Chapter 22-73
• During protein synthesis amino acids do not directly
•
•
•
•
•
interact with the codons of an mRNA molecule.
tRNA molecules as intermediaries deliver amino
acids to mRNA.
Two important features of the tRNA structure
The 3’ end of tRNA is where an amino acid is
covalently bonded to the tRNA.
The loop opposite to the open end of tRNA is the site
for a sequence of three bases called an anticodon.
Anticodon - a three-nucleotide sequence on a tRNA
molecule that is complementary to a codon on an
mRNA molecule.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-74
• Translation – a process in which mRNA
codons are deciphered to synthesize a
protein molecule
• Ribosome – an rRNA–protein complex serves as the site of protein synthesis:
• Contains 4 rRNA molecules and ~80 proteins packed into two rRNA-protein subunits (one small
and one large)
• ~65% rRNA and 35% protein by mass
• A ribosome’s active site – Large subunit
• Ribosome is a RNA catalyst
• The mRNA binds to the small subunit of the
ribosome.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-75
Five Steps of Translation Process
• Activation of tRNA: addition of specific amino acids
•
•
•
•
to the 3’-OH group of tRNA.
Initiation of protein synthesis: Begins with binding of
mRNA to small ribosomal subunit such that its first
codon (initiating codon AUG) occupies a site called
the P site (peptidyl site)
Elongation: Adjacent to the P site in an mRNA–
ribosome complex is A site (aminoacyl site) and the
next tRNA with the appropriate anticodon binds to it.
Termination: The polypeptide continues to grow via
translocation until all necessary amino acids are in
place and bonded to each other.
Post-translational processing – gives the protein the
final form it needs to be fully functional
Chemistry 121 Winter 2010 LA Tech
Chapter 22-76
Efficiency of mRNA Utilization
• Polysome (polyribosome): complex of mRNA
and several ribosomes
• Many ribosomes can move simultaneously
along a single mRNA molecule
• The multiple use of mRNA molecules reduces
the amount of resources and energy that the
cell expends to synthesize needed protein.
• In the process – several ribosomes bind to a
single mRNA - polysomes.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-77
Mutation
• An error in base sequence reproduced
during DNA replication
• Errors in genetic information is passed on
during transcription.
• The altered information can cause changes
in amino acid sequence during protein
synthesis and thereby alter protein function
• Such changes have a profound effect on an
organism.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-78
Mutagens
• Mutations are caused by mutagens
• A mutagen is a substance or agent that causes a
change in the structure of a gene:
• Radiation and chemical agents are two important
types of mutagens
• Ultraviolet, X-ray, radioactivity and cosmic
radiation are mutagenic –cause cancers
• Chemical agents can also have mutagenic effects
• E.g., HNO2 can convert cytosine to uracil
• Nitrites, nitrates, and nitrosamines – can form
nitrous acid in cells
• Under normal conditions mutations are repaired by
repair enzymes
Chemistry 121 Winter 2010 LA Tech
Chapter 22-79
Viruses
• Viruses: Tiny disease causing agents with
outer protein envelope and inner nucleic acid
core
• They can not reproduce outside their host
cells (living organisms)
• Invade their host cells to reproduce and in
the process disrupt the normal cell’s
operation
• Virus invade bacteria, plants animals, and
humans:
• Many human diseases are of viral origin, e. g. Common
cold, smallpox, rabies, influenza, hepatitis, and AIDS
Chemistry 121 Winter 2010 LA Tech
Chapter 22-80
Vaccines
• Inactive virus or bacterial envelope
• Antibodies produced against inactive viral or
bacterial envelopes will kill the active
bacteria and viruses
Chemistry 121 Winter 2010 LA Tech
Chapter 22-81
Viruses
• Viruses attach to the host cell on the outside
cell surface and proteins of virus envelope
catalyze the breakdown of the cell membrane
and forms a hole
• Viruses then inject their DNA or RNA into the
host cell
• The viral genome is replicated, proteins
coding for the viral envelope are produced in
hundreds of copies.
• Hundreds of new viruses are produced using
the host cell replicated genome and proteins
in short time
Chemistry 121 Winter 2010 LA Tech
Chapter 22-82
• DNA molecules that have been synthesized by
splicing a sequence of segment DNA (usually a
gene) from one organism to the DNA of another
organism
• Genetic Engineering (Biotechnology):
• The study of biochemical techniques that allow the transfer
of a “foreign” gene to a host organism and produce the
protein associated with the added gene
• Bacterial strains such as E. coli inserted with circular
plasmids, and/or yeast cells carrying vectors containing
foreign genes are used for this purpose
• Plasmids (double stranded DNA) replicate independently in
bacteria or yeast
Chemistry 121 Winter 2010 LA Tech
Chapter 22-83
Recombinant DNA Production using a Bacterial Plasmid
•
•
•
•
•
•
Dissolution of cells:
• E. coli cells of a specific strain containing the plasmid of interest are
treated with chemicals to dissolve their membranes and release the
cellular contents
Isolation of plasmid fraction:
• The cellular contents are fractionated to obtain plasmids
Cleavage of plasmid DNA:
• Restriction enzymes are used to cleave the double-stranded DNA
Gene removal from another organism:
• Using the same restriction enzyme the gene of interest is removed
from a chromosome of another organism
Gene–plasmid splicing:
• The gene (from Step 4) and the opened plasmid (from Step 3) are
mixed in the presence of the enzyme DNA ligase to splice them
together.
Uptake of recombinant DNA:
• The recombinant DNA prepared in stept 5 are transferred to a live E.
coli culture where they can be replicated, trasncribed and translated.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-84
Clones
• Transformed cell can reproduce a large
number of identical cells –clones:
• Clones are the cells that have descended from a
single cell and have identical DNA
• Given bacteria grow very fast, within few
hours 1000s of clones will be produced
• Each clone can synthesize the protein
directed by foreign gene it carries
Chemistry 121 Winter 2010 LA Tech
Chapter 22-85
The polymerase chain reaction (PCR)
•
•
•
The polymerase chain reaction (PCR) is a method for rapidly
producing multiple copies of a DNA nucleotide sequence
(gene).
This method allows to produce billions of copies of a specific
gene in a few hours.
PCR is very easy to carryout and the requirements are:
• Source of gene to be copied
• Thermostabel DNA polymerase
• Deoxynucleotide triphosphates (dATP, dGTP, dCTP and
dTTP)
• A set of two oligonucleotides with complementary
sequence to the gene (primers)
• Thermostable plastic container and
• Source of heat
Chemistry 121 Winter 2010 LA Tech
Chapter 22-86
DNA sequencing
• DNA sequencing is a method by which the
base sequence in a DNA molecule (or a
portion of it) is determined.
• Discovered in 1977 by Fredrick Sanger
• Concept in DNA sequencing:
• Selective interruption of polynucleotide
synthesis using 2’,3’-dideoxyribonucleotide
triphosphates (ddNTPs).
Chemistry 121 Winter 2010 LA Tech
Chapter 22-87
ddNTPs Fragments
• This interruption of synthesis leads to the
formation of every possible nucleotide site
mixture.
• These nucleotides are labeled using
radioactive dNTP during their synthesis.
• The radiolablled nucleotides are then
separated on a gel by electrophoresis
Chemistry 121 Winter 2010 LA Tech
Chapter 22-88
Basic steps involved in DNA
sequencing
• Step 1: Cleavage of DNA using restriction enzymes:
Restriction enzymes are used to cleave the large
DNA molecule into smaller fragments (100–200 base
pairs).
• Step 2: Separation into individual components: The
mixture of small DNA fragments generated by the
restriction enzymes is separated into individual
components via gel electrophoresis techniques.
• Step 3: Separation into single strands: A given DNA
fragment is separated into its two strands by
chemical methods to use it as a template in step 4.
Chemistry 121 Winter 2010 LA Tech
Chapter 22-89
Basic steps involved in DNA
sequencing
Chemistry 121 Winter 2010 LA Tech
Chapter 22-90